Epithelial sodium channel (ENaC) activity in the collecting duct is essential for maintaining extracellular fluid volume and blood pressure. Serum and glucocorticoid regulated kinase 1 (sgk1) has emerged as a key component of the aldosterone signaling pathway that stimulates ENaC in the collecting duct. The most studied mechanism by which sgk1 increases ENaC activity involves an increase in the number of channels in the apical cell membrane. However, our laboratory has recently demonstrated that sgk1 can also stimulate the open probability (Po) of ENaC, which represents a novel means by which sgk1 acts. There remain critical gaps in our knowledge about the mechanisms that underlie sgk1 regulation of ENaC, including questions about: 1) how does sgk1 stimulate ENaC Po, and 2) whether this mode of ENaC regulation is physiologically important. The objective of the proposed research is to define novel mechanisms by which sgk1 stimulates ENaC Po in the collecting duct and determine whether this mode of ENaC regulation is important in vivo. We hypothesize that sgk1 interacts with and phosphorylates ENaC to stimulate channel Po. This mechanism of sgk1 action is distinct from sgk1-mediated regulation of Nedd4-2, a major regulator of cell surface ENaC expression. We propose three Specific Aims. In Aim 1, we will test whether sgk1 stimulates ENaC Po independent of Nedd4-2. We will introduce mutations in sgk1 or ENaC that: 1) disrupt Nedd4-2-mediated regulation of ENaC; or 2) disrupt ENaC gating. We will then test whether sgk1 can still increase channel Po under these conditions. We will also test whether sgk1 can stimulate ENaC Po in split-open collecting ducts from Nedd4-2 knockout mice. In Aim 2, we will test whether sgk1 stimulates ENaC Po through channel phosphorylation. We will use site- directed mutagenesis to identify sites in ENaC that are phosphorylated by sgk1. We will then test whether sgk1 can stimulate Po of these mutant channels. In Aim 3, we will test whether sgk1 stimulates ENaC Po through channel interaction. We will use site-directed mutagenesis and blocking peptides to identify sites in sgk1 responsible for mediating interaction with ENaC. We will use these sgk1 mutants and cell-permeant peptides to disrupt the sgk1-ENaC interaction and test whether these reagents will impair sgk1-mediated stimulation of ENaC Po and activity in collecting duct cells and split-open collecting ducts from wild type mice. The proposed research is expected to expand the current paradigm of sgk1 action and provide novel insights into how sgk1 stimulates ENaC Po in the collecting duct. We expect that this research will delineate the mechanisms by which sgk1 stimulates ENaC Po and establish the physiological importance of this mode of ENaC regulation in vivo. The impact of the proposed research may provide new insights into the pathogenesis of hypertension and suggest new targets (e.g. sgk1-ENaC interaction) for therapeutic intervention.